WO2021067740A1 - A crnn loss of function rodent model - Google Patents
A crnn loss of function rodent model Download PDFInfo
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
- A01K2217/056—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to mutation of coding region of the transgene (dominant negative)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
Definitions
- This disclosure relates to a genetically modified rodent and use thereof as a rodent model. More specifically, this disclosure relates to rodent (e.g., mouse or rat) comprising a loss of function mutation in an endogenous Crnn (cornulin) gene, and to use of such a rodent animal as a rodent model of skin inflammation disorders (e.g., psoriasis).
- rodent e.g., mouse or rat
- Crnn cornulin
- rodents e.g., mice and rats
- isolated rodent cells e.g., ES cells
- compositions e.g., targeting vectors
- a rodent whose genome comprises a loss of function mutation in an endogenous Crnn gene at an endogenous rodent Crnn locus.
- a loss of function mutation in an endogenous Crnn gene at an endogenous rodent Crnn locus results in the lack of a functional Crnn polypeptide being expressed from the Crnn locus.
- a loss of function mutation comprises a deletion, in whole or in part, of the coding sequence of an endogenous rodent Crnn gene.
- the deletion comprises exon 2 in whole or in part, and/or exon 3 in whole or in part, of an endogenous rodent Crnn gene.
- the deletion comprises a nucleic acid sequence from the nucleotide after the ATG start codon in exon 2 through the stop codon of an endogenous rodent Crnn gene.
- the rodent Crnn locus which comprises a loss of function mutation in an endogenous Crnn gene further comprises a reporter gene.
- the reporter gene is operably linked to the endogenous Crnn promoter at the Crnn locus.
- the Crnn locus comprises a deletion beginning from the nucleotide after the ATG start codon in exon 2 through the stop codon of the endogenous rodent Crnn gene, and comprises a reporter gene coding sequence that is fused in-frame to the start (ATG) codon of the Crnn locus.
- the reporter gene is lacZ.
- the reporter gene is selected the group consisting of luciferase, green fluorescent protein (GFP), enhanced GFP (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, and MmGFP.
- a rodent is homozygous for a loss of function mutation in an endogenous Crnn gene. In some embodiments, a rodent is heterozygous for a loss of function mutation in an endogenous Crnn gene.
- a rodent is a mouse. In some embodiments, a rodent is a rat.
- a progeny of any of the rodents disclosed herein is also provided herein.
- disclosed herein is an isolated rodent cell or tissue whose genome comprises a loss of function mutation in an endogenous rodent Crnn gene at an endogenous rodent Crnn locus.
- the isolated rodent cell is a rodent embryonic stem cell, or a rodent egg.
- a rodent embryo whose genome comprises a loss of function mutation in an endogenous rodent Crnn gene at an endogenous rodent Crnn locus.
- a method of making a rodent whose genome comprises a loss of function mutation in an endogenous rodent Crnn gene at an endogenous rodent Crnn locus.
- a rodent genome is modified by introducing a nucleic acid molecule into a rodent embryonic stem cell, which nucleic acid molecule comprises an insert nucleotide sequence to be integrated into the endogenous rodent Crnn locus, flanked by polynucleotide sequences that are homologous to nucleic acid sequences at the endogenous rodent Crnn locus.
- a loss of function mutation comprises a deletion, in whole or in part, of the coding sequence of an endogenous rodent Crnn gene.
- the deletion comprises a nucleic acid sequence from the nucleotide after the ATG start codon in exon 2 through the stop codon of an endogenous rodent Crnn gene.
- a rodent made by the present method further comprises a reporter gene.
- a reporter gene can be included in the nucleic acid sequence being introduced into the genome of a rodent embryonic stem cell.
- the reporter gene is operably linked to the endogenous Crnn promoter at the Crnn locus in the modified genome.
- the Crnn locus of a modified genome comprises a deletion beginning from the nucleotide after the ATG start codon in exon 2 through the stop codon in exon 3 of the endogenous rodent Crnn gene, and comprises a reporter gene coding sequence that is fused in-frame to the start (ATG) codon of the Crnn locus.
- the reporter gene is lacZ.
- the reporter gene is selected the group consisting of luciferase, green fluorescent protein (GFP), enhanced GFP (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, and MmGFP.
- GFP green fluorescent protein
- eGFP enhanced GFP
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- eYFP enhanced yellow fluorescent protein
- BFP blue fluorescent protein
- eBFP enhanced blue fluorescent protein
- DsRed and MmGFP.
- MmGFP MmGFP.
- a rodent made by the present method further comprises a selection marker.
- a selection marker gene can be included in the nucleic acid sequence being introduced into the genome of a rodent embryonic stem cell.
- the nucleic acid sequence may further comprise site-specific recombinase recognition sites flanking the selection marker gene, which site-specific recombinase recognition sites are oriented to direct an excision of the selection marker by a recombinase.
- a rodent made by the present method is heterozygous for a loss of function mutation in an endogenous Crnn gene. Rodents heterozygous for a loss of function mutation in an endogenous Crnn gene can be bred with each other to obtain rodents homozygous for the loss of function mutation in an endogenous Crnn gene.
- a rodent made by the present method is a mouse.
- a rodent made by the present method is a rat.
- a targeting nucleic acid construct comprising a nucleotide sequence to be integrated into a rodent Crnn locus, flanked by a 5' nucleotide sequence and a 3' nucleotide sequence that are homologous to nucleotide sequences at the rodent Crnn locus, wherein integration of the nucleotide sequence into the rodent Crnn locus results in a loss of function mutation in the endogenous rodent Crnn gene as described herein.
- the targeting nucleic acid construct can be designed so as to be capable of integrating the n nucleotide sequence into a mouse or rat Crnn gene at an endogenous mouse or rat Crnn locus.
- the nucleotide sequence to be integrated into a rodent Crnn gene at an endogenous rodent Crnn locus includes a reporter gene.
- the nucleotide sequence to be integrated into a rodent Crnn gene at an endogenous rodent Crnn locus includes a selectable marker gene.
- a method of breeding comprising breeding a first rodent whose genome comprises a loss of function mutation in an endogenous rodent Crnn gene, with a second rodent, resulting in a progeny rodent whose genome comprises the loss of function mutation in an endogenous rodent Crnn gene.
- a rodent whose genome comprises a loss of function mutation in an endogenous Crnn gene as an animal model for human disorders such as skin inflammation (e.g., psoriasis).
- a rodent model of skin inflammation diseases comprises a rodent disclosed herein, to which a skin inflammation inducing agent is applied.
- skin inflammation is induced by imiquimod (IMQ).
- IMQ imiquimod
- a rodent disclosed herein displays enhanced inflammation in an experimentally induced skin inflammation model as compared to a wild type rodent.
- a method of assessing the therapeutic efficacy of a candidate compound for treating such as skin inflammation comprising administering an agent to a rodent disclosed herein to induce skin inflammation, administering a candidate compound to the rodent, and determining whether the candidate compound inhibits and/or reduces induced skin inflammation.
- the agent administered to induce skin inflammation is IMQ.
- a candidate compound is administered to the rodent before, during, or after the administration of an agent that induces inflammation.
- a candidate compound can be a small molecule compound, a nucleic acid inhibitor, or an antigen-binding protein such as an antibody.
- FIG.1 depicts an exemplary targeting strategy for ablation of the Crnn gene and generation of a Crnn knock-out (Crnn -/- ) mouse.
- FIGS.2A-2G IMQ-induced psoriasis reveals barrier defects in Crnn-/- mice.
- 2A Experimental setup of IMQ treatment and skin collection.
- 2B Representative photographs of vehicle or IMQ-treated Crnn-/-, +/-, and WT littermates.
- 2C Clinical scores and 2D) Trans-epidermal water loss (TEWL) of vehicle or IMQ-treated Crnn-/-, +/-, and WT littermates prior to skin harvest.
- 2E Immunofluorescence of Krt10, Krt5, Integrin B4, and DAPI in skin harvested from vehicle or IMQ-treated Crnn-/- and WT littermates. Bar, 100uM. Quantification of 2E) including average thickness 2F) and average number of nuclei retained 2G) in basal to spinous layers per 250uM tissue.
- FIGS.3A-3I Crnn-/- mice display increased IMQ-induced skin inflammation.
- 3A Hematoxylin & Eosin-stained sections of skin from vehicle or IMQ-treated Crnn-/-, and WT littermates.
- 3B Total pathology score of skin from vehicle or IMQ-treated IMQ-treated Crnn-/-, +/-, and WT littermates comprised of a score (0-within normal limits; 1-minimal; 2-mild; 3-moderate; 4- severe) from seven parameters including 3C) acanthosis, 3D) Munro’s microabscess, and 3E) parakeratosis with hypogranulosis, 3F) Myeloperoxidase 3G) TNF ⁇ 3H) KC/GRO and 3I) Il-1ß levels in skin harvested from vehicle or IMQ-treated Crnn-/-, +/-, and WT littermates.
- Crnn knock-out mice also referred to as "Crnn KO” or “Crnn-/-” mice, and Crnn+/- mice
- Phenotyping of the Crnn KO mice shows strong expression of a reporter gene in the epidermis consistent with the known function of Crnn as a member of the epidermal differentiation complex (EDC).
- EDC epidermal differentiation complex
- the Crnn-/- and Crnn+/-mice have been shown to be more susceptible to the IMQ treatment as compared to wild type mice.
- the IMQ-treated Crnn-/-and Crnn+/-mice showed increased skin thickness and inflammation and elevation of pro-inflammatory cytokines versus IMQ-treated wild- type mice.
- the data on the phenotypic characterization of the Crnn loss of function mouse model supports the notion that deficiency of Cornulin in the epidermis increases the susceptibility to developing psoriasis.
- CRNN (Cornulin) is a member of the epidermal differentiation complex (EDC). It contains two EF-hand Ca2+ binding domains in its N-terminus and two glutamine- and threonine-rich 60 amino acid repeats in its C-terminus. EDC proteins are crucial for development, maintenance and maturation of the epidermis. [0039] The protein structure and the genomic structure of Cornulin are conserved across species. Exemplary wild type Crnn mRNA and protein sequences from human, mouse and rat are available in GenBank under the following accession numbers, and are also set forth as SEQ ID NOS: 1-6 in the Sequence Listing.
- mutation includes an addition, deletion, or substitution of one or more nucleotides in a gene (e.g., a wild type Crnn allele).
- a mutation is a substitution of a single nucleotide.
- a mutation is a deletion of one or more nucleotides, e.g., one or more nucleotides in the coding sequence of a gene.
- a loss of function mutation in a gene includes a deletion of a contiguous nucleic acid sequence, e.g., one or more exons, in full or in part, of a gene.
- a mutation in a gene results in an addition, deletion, or substitution of one or more amino acids in the encoded protein (e.g., a wild type Crnn protein).
- a mutation in a gene results in a lack of functional protein being expressed or produced - such a mutation is also referred to herein as a "loss of function" mutation.
- a loss of function mutation in a gene causes a deletion of one or more amino acids, resulting in a mutant protein that lacks the function of the wild type protein.
- a loss of function mutation in a gene includes a deletion of a contiguous nucleic acid sequence, e.g., one or more exons in full or in part, resulting in a lack of expression of a protein from the mutant gene or resulting in an expression of a protein that lacks the function of the wild type protein.
- a loss of function mutation in a Crnn gene includes a deletion of the first coding exon (i.e., exon 2) in whole or in part, e.g., the coding portion of exon 2 beginning from the nucleotide after the ATG codon.
- a loss of function mutation in a Crnn gene includes a deletion of the second coding exon (i.e., exon 3) in whole or in part.
- a loss of function mutation in a Crnn gene includes a deletion of a nucleotide sequence coding for a peptide portion that comprises an EF-hand domain, or amino acids involved in Ca2+ binding, and/or the S-100 domain.
- a loss of function mutation in a Crnn gene includes a deletion of the coding sequence of exon 2 beginning from the nucleotide after the ATG codon and a deletion of exon 3 at least through the stop codon. In some embodiments, a loss of function mutation in a Crnn gene includes a deletion of the coding sequence of exon 2 beginning from the nucleotide after the ATG codon and a deletion of entire exon 3 (i.e., through the stop codon and the 3' untranslated region).
- a rodent whose genome comprises a loss of function mutation in an endogenous Crnn gene further comprises an insertion of a reporter gene, and wherein the reporter gene is operably linked to the endogenous rodent Crnn promoter at the endogenous rodent Crnn locus.
- a genomic fragment beginning from the nucleotide after the start codon in exon 2 through the whole or part of exon 3 of an endogenous Crnn gene has been deleted, and the reporter gene is inserted immediately downstream of the start codon of the endogenous rodent Crnn gene. In such linkage, expression of the reporter gene is expected to resemble the expression pattern of an unmodified endogenous rodent Crnn gene.
- the reporter gene is a LacZ gene.
- the reporter gene is a gene encoding a protein selected the group consisting of luciferase, green fluorescent protein (GFP), enhanced GFP (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, and MmGFP.
- GFP green fluorescent protein
- eGFP enhanced GFP
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- eYFP enhanced yellow fluorescent protein
- BFP blue fluorescent protein
- eBFP enhanced blue fluorescent protein
- DsRed and MmGFP
- the rodent is a mouse of a C57BL strain, for example, a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
- a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
- the rodent is a mouse of a 129 strain, for example, a 129 strain selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999), Mammalian Genome 10:836; Auerbach et al.
- a 129 strain selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/S
- the rodent is a mouse that is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain.
- the mouse is a mix (i.e., hybrid) of aforementioned 129 strains, or a mix of aforementioned C57BL strains, or a mix of a C57BL strain and a 129 strain.
- the mouse is a mix of a C57BL/6 strain with a 129 strain.
- the mouse is a VGF1 strain, also known as F1H4, which is a hybrid of C57BL/6 and 129.
- the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another aforementioned strain.
- the rodent is a rat. In certain embodiments, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In other embodiments, the rat is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
- a genetically modified rodent having a loss of function mutation in the endogenous Crnn gene is more susceptible to an imiquimod (IMQ) treatment (e.g., by displaying increased inflammation) as compared to a wild type rodent in an IMQ-induced model of psoriasis.
- IMQ is applied topically to the skin of a rodent to induce skin inflammation.
- IMQ is provided in a carrier suitable for topical application, e.g., a cream, a gel, including commercially available IMQ creams (e.g., such as those from Aldara).
- IMQ is applied to the rodent skin daily at a daily dose of 1 to 5 mg, 2 to 4 mg, or 3 to 3.5 mg, for a period of 3 to 5 days, e.g., for 4 days.
- a daily topical application at a daily dosage of about 3.125 mg for 4 days is suitable for inducing acute skin inflammation.
- the severity of the skin inflammation can be evaluated by (i) using an adapted version of the clinical Psoriasis Area and Severity Index based on measuring and independently scoring erythema, scaling and thickening of the skin; (ii) performing histopathological analysis of skin tissues, e.g., to evaluate and score each of the following parameters: hyperkeratosis, parakeratosis, Munro’s microabscess (accumulation of neutrophils in the stratum corneum), acanthosis, epidermal erosion or ulceration, inflammation (inflammatory cell infiltrates) in the dermis and hypodermis, blood vessel congestion in the dermis and hypodermis, and to determine a total pathology score; (iii) measuring concentrations of proinflammatory cytokines in the skin homogenates, including e.g., concentrations of myeloperoxidase, KC-GRO, IL-6, IL-1b, TNFa, IL-
- a genetically modified rodent having a loss of function mutation in an endogenous Crnn gene displays increased skin inflammation after an IMQ treatment as compared to wild-type mice based on visual scoring, individual scoring or total scoring, of erythema, scaling and thickening.
- a genetically modified rodent having a loss of function mutation in an endogenous Crnn gene displays increased skin inflammation after an IMQ treatment as compared to wild-type mice based on measured skin thickness.
- a genetically modified rodent having a loss of function mutation in an endogenous Crnn gene displays increased skin inflammation after an IMQ treatment as compared to wild-type mice based on an increased total score from evaluating and scoring each of the following parameters: hyperkeratosis, parakeratosis, Munro’s microabscess (accumulation of neutrophils in the stratum corneum), acanthosis, epidermal erosion or ulceration, inflammation (inflammatory cell infiltrates) in the dermis and hypodermis, blood vessel congestion in the dermis and hypodermis.
- a genetically modified rodent having a loss of function mutation in an endogenous Crnn gene displays increased skin inflammation after an IMQ treatment as compared to wild-type mice based on elevated levels of pro-inflammatory cytokines (e.g., one or more of myeloperoxidase, KC-GRO, IL-6, IL-1b, TNFa, IL-36 ligands such as IL-36a and IL-36b) in skin homogenates by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.
- pro-inflammatory cytokines e.g., one or more of myeloperoxidase, KC-GRO, IL-6, IL-1b, TNFa, IL-36 ligands such as IL-36a and IL-36b
- a genetically modified rodent having a loss of function mutation in an endogenous Crnn gene displays an increased trans-epidermal water vapor loss (TEWL) after an IMQ treatment as compared to wild-type mice, e.g., an increase by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.
- TEWL trans-epidermal water vapor loss
- isolated rodent cells or tissues whose genome comprises a loss of function mutation in an endogenous Crnn gene, described herein.
- an isolate rodent cell is an embryonic stem (ES) cell. Rodent embryos and eggs comprising a loss of function mutation in an endogenous Crnn gene are also provided.
- the method comprises modifying a rodent genome such that the modified rodent genome comprises a loss of function mutation in an endogenous rodent Crnn gene at an endogenous rodent Crnn locus, and obtaining a rodent comprising the modified genome.
- a rodent genome is modified by, e.g., employing a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Cas protein (i.e., a CRISPR/Cas system), such that the modified genome includes a loss of function mutation in an endogenous rodent Crnn gene at an endogenous rodent Crnn locus.
- ZFN zinc finger nuclease
- TALEN transcription activator-like effector nuclease
- Cas protein i.e., a CRISPR/Cas system
- a rodent genome is modified by introducing a nucleic acid molecule into a rodent embryonic stem (ES) cell, wherein the nucleic acid molecule comprises a nucleotide sequence desired to be integrated into the Crnn locus (i.e., an insert nucleotide sequence) to create a loss of function mutation in an endogenous rodent Crnn gene.
- ES rodent embryonic stem
- the insert nucleotide sequence is flanked by polynucleotide sequences that are homologous to nucleic acid sequences at the endogenous rodent Crnn locus and capable of mediating homologous recombination of the insert nucleotide sequence into the genome of the ES cell, such that the modified genome comprises a loss of function mutation in an endogenous rodent Crnn gene at the endogenous rodent Crnn locus.
- the resulting genetically modified rodent embryonic stem cell can then be used in making a genetically modified rodent.
- the insert nucleotide sequence to be integrated into the genome of a rodent ES cell is provided in a targeting nucleic acid construct (i.e., a targeting vector), preferably a DNA vector.
- a targeting nucleic acid construct i.e., a targeting vector
- the insert nucleotide sequence also contains a selectable marker gene (e.g., a self deleting cassette containing a selectable marker gene, as described in U.S. Patent Nos.8,697,851, 8,518,392 and 8,354,389, all of which are incorporated herein by reference), which can be flanked by or comprises site-specific recombination sites (e.g., loxP, Frt, etc.).
- the selectable marker gene can be placed on the vector adjacent to the mutation to permit easy selection of transfectants.
- the insert nucleotide sequence also contains a reporter gene.
- a targeting vector e.g., a BAC vector
- ES rodent embryonic stem
- Homologous recombination in recipient cells can be facilitated by introducing a break in the chromosomal DNA at the integration site, which may be accomplished by targeting certain nucleases to the specific site of integration.
- DNA-binding proteins that recognize DNA sequences at the target locus are known in the art.
- ZFNs zinc finger nucleases
- TALENs Transcription activator-like (TAL) effector nucleases
- RGENs RNA-guided endonucleases
- a targeting vector carrying a nucleic acid of interest (e.g., an insert nucleotide sequence for generating a loss of function Crnn mutant gene), flanked by 5' and 3' homology arms, is introduced into a cell with one or more additional vectors or mRNA.
- the one or more additional vectors or mRNA contain a nucleotide sequence encoding a site-specific nuclease, including but not limited to a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, and an RNA-guided DNA endonuclease.
- ZFN zinc finger nuclease
- TALEN transcription activator-like effector nuclease
- TAL effector domain fusion protein e.
- ES cells having an insert nucleotide sequence integrated in the genome can be selected. After selection, positive ES clones can be modified, e.g., to remove a self- deleting cassette, if desired. ES cells having the mutation integrated in the genome can then be used as donor ES cells for injection into a pre-morula stage embryo (e.g., 8-cell stage embryo) by using the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US 2008/0078000 A1), or methods described in US 2014/0235933 A1 and US 2014/0310828 A1.
- a pre-morula stage embryo e.g., 8-cell stage embryo
- Rodent pups bearing the mutant allele can be identified by genotyping of DNA isolated from tail snips using a modification of allele (MOA) assay (Valenzuela et al., supra) that detects the presence of the mutant sequence or a selectable marker gene.
- MOA modification of allele
- a method which comprises breeding a first genetically modified rodent as described hereinabove (e.g., a rodent whose genome comprises a loss of function Crnn mutation at an endogenous rodent Crnn locus), with a second rodent, resulting in a progeny rodent whose genome comprises the loss of function Crnn mutation.
- the progeny may possess other desirable phenotypes or genetic modifications inherited from the second rodent used in the breeding.
- the progeny rodent is heterozygous for the loss of function Crnn mutation.
- the progeny rodent is homozygous for the loss of function Crnn mutation.
- a progeny rodent whose genome comprises a loss of function Crnn mutation at an endogenous rodent Crnn locus, wherein the progeny rodent is produced by a method comprising breeding a first genetically modified rodent as described hereinabove (e.g., a rodent whose genome comprises a loss of function Crnn mutation at an endogenous rodent Crnn locus), with a second rodent.
- the progeny rodent is heterozygous for the loss of function Crnn mutation.
- the progeny rodent is homozygous for the a loss of function Crnn mutation.
- rodent Model and Use Thereof is use of a rodent whose genome comprises a loss of function mutation in an endogenous Crnn gene as an animal model of diseases, including skin inflammation disorders such as psoriasis. Such model is useful for understanding skin inflammation biology and processes, as well as testing, screening, or identifying an agent that treats skin inflammation. [0071] In some embodiments, disclosed here are methods for testing, screening, or identifying an agent useful for treating skin inflammation.
- compounds that can be evaluated using the rodents disclosed include, for example, small molecule compounds, nucleic acid-based compounds (e.g., siRNA, ribozyme, antisense construct, etc.), and an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof).
- Candidate compounds can be evaluated by inducing skin inflammation, e.g., by topical application of IMQ, in a rodent disclosed herein, and determining whether a candidate compound can treat or inhibit the induced inflammation.
- the term "treating” or “inhibiting” includes ameliorating the severity, slowing down the progression, eliminating, delaying or preventing the onset of the induced inflammation and symptoms, or a combination thereof.
- a rodent is administered with a candidate compound prior to, during, together with, or after administration of an agent (e.g., IMQ) that induces skin inflammation.
- Candidate compounds may be dosed via any desired route of administration including parenteral and non-parenteral routes of administration.
- Parenteral routes include, e.g., intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebro ventricular, intracranial, intrapleural or other routes of injection.
- Non-parenteral routes include, e.g., oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular.
- Administration may also be by continuous infusion, local administration, sustained release from implants (gels, membranes or the like), and/or intravenous injection.
- a candidate compound is administered subcutaneously at or near the skin area where an agent (e.g., IMQ) that induces skin inflammation is applied.
- an agent e.g., IMQ
- a compound is considered to be effective if it inhibits skin inflammation as compared to appropriate control rodents.
- suitable control rodents can include, e.g., genetically modified rodents comprising the same loss of function mutation but without being subjected to an induced inflammation treatment; genetically modified rodents comprising the same loss of function mutation which have been subjected to an induced inflammation treatment without any candidate compound or with a control compound not expected to have any therapeutic efficacy; and genetically modified rodents comprising the same loss of function mutation which have been subjected to an induced inflammation treatment and a compound known to be therapeutically effective.
- the present description is further illustrated by the following examples, which should not be construed as limiting in any way.
- C57BL/6NTac embryonic stem cells were targeted for ablation of a portion of the Crnn locus, beginning just after the start ATG and ending 241 base pairs beyond the stop codon.
- a LacZ reporter module was inserted in frame with the Crnn start, followed by a fLoxed neomycin resistance cassette for selection of correctly targeted ESCs. See FIG.1.
- Correctly targeted ESCs were microinjected into 8-cell embryos from Charles River Laboratories Swiss Webster albino mice, yielding F0 VelociMice® that were 100% derived from the targeted cells (Poueymirou et al.2007, supra).
- mice were subsequently bred to homozygosity and maintained in the Regeneron animal facility during the study period.
- the resistance cassette was removed in the F0 germline using self-deleting technology, leaving a single loxP.
- Crnn-/+ heterozygous mice and C57Bl/6NTac wildtype littermates were also included in various analysis.
- Example 2. LacZ Expression in Crnn-/- Mice [0078] Six to 8-week old mice were deeply anesthetized via Ketamine/Xylazine (120/5 mg/kg) IP injection and fixed by cardiac perfusion using a 0.2% glutaraldehyde, 4% paraformaldehyde solution.
- a daily topical dose of 62.5mg of Aldara translated into a daily dose of 3.125 mg of an active compound. Two or three days after the treatments, the back skin of mice started to display signs of erythema, scaling and thickening.
- TEWL Trans-Epidermal Water Loss
- mice were anesthetized in an isoflurane inhalation chamber (3% isofluorane for 80 seconds) before the VapoMeter® (equipped with its small adaptor) was applied to the back skin of the mice to take the reading. Baseline readings were taken on the first day of treatment, prior to IMQ application, and again 24h after the 4th IMQ treatment, just prior to euthanasia.
- Immunofluorescence (IF) Assays [0082] For IF assays, back skin biopsies were collected post euthanasia, backed with nitrocellulose, embedded in cryomatrix (Tissue-Tek® O.C.T. Compound, Electron Microscopy Sciences) and stored at -80°C until use.
- the frozen tissue blocks were sectioned at a thickness of 12mm on a cryostat (Leica), and collected on microscope slides (VWR® Superfrost®). Slides were allowed to dry at room temperature, and then fixed in 4% PFA in PBS 1x for 10 minutes. Slides were rinsed 5 times with 1x PBS and then blocked for 1h at room temperature in the following blocking buffer: 5% normal donkey serum, 0.5% bovine serum albumin, 2.5% fish gelatin and 0.3% Triton X-100 in PBS. The slides were incubated with primary antibodies diluted in blocking solution at 4°C overnight, washed three times with PBS and incubated with secondary antibodies in blocking solution at room temperature for 1 hour.
- Histology and pathology scoring were performed by a licensed veterinarian. Samples were processed, embedded in paraffin, and sectioned at 5 ⁇ m. Hematoxylin & Eosin (H&E) staining was performed on a BOND RX autostainer (Leica Biosystems) using standard protocols. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma® and Coverslipper film (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 apparatus (Leica Biosystems).
- Hematoxylin & Eosin- stained sections of skin from 6-8 week-old female vehicle or IMQ-treated Crnn-/-, +/-, and WT littermates were examined for histomorphological changes related to psoriasis, by implementing semi-quantitative grading scheme that took into account the following seven parameters (Nakajima, K. and Sano, S. (2016), J Dermatol, 45: 252-263.
- IMQ-induced psoriasis reveals epidermal barrier defects in Crnn-/- mice.
- IMQ imiquimod
- TLR7 toll like receptor 7
- Trans-epidermal water loss (TEWL), a measure of epidermal barrier integrity, measured according to manufacturer’s instructions using a VapoMeter®, was greatest in Crnn-/- mice 24h after the final IMQ application (FIG.2D).
- average thickness was significantly increased in IMQ-treated Crnn-/- mice, and was accompanied by a significantly higher retention of nuclei compared to WT controls (FIGS.2F-G).
- Acanthosis (FIG.3C); Munro’s microabscess, (FIG.
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